It remains difficult to manipulate protein kinase activity with precise timing and localization in living cells. Furthermore, targeted manipulation of kinase activity only in selected protein complexes is currently impossible for the majority of biological studies. We have recently developed a new generally applicable method for rapamycin-regulated (RapR) activation of kinases and successfully applied it to three kinases from two different classes, tyr and ser/thr kinases (FAK, Src, and p38). Here, we propose to employ RapR technology to develop new broadly applicable methods for selective regulation of highly homologous kinases in living cells, and targeted activation of kinases only when they are in specific protein complexes. We will also achieve light- mediated localized regulation of kinases using caged rapamycin. These methods will be applied to identify the roles of different Src family kinases. These highly homologous kinases serve as a good test of the specificity of the new approaches and will provide new capabilities to answer previously intractable questions. Localized activation will be used to probe the spatio-temporal regulation of pathways modulating cell protrusion and polarization.